Project Summary Intracellular transport is ubiquitous in animal cells and has fundamental importance for diverse biological phenomena, such as secretion, neuronal signaling, organization of endomembranes, and mitosis. The driving force for intracellular transport is provided by molecular motors bound to the surface of cargo organelles and moving along microtubules (MTs) and actin filaments (AFs). Transport of specific organelles is generally driven by several types of molecular motors simultaneously bound to their surface. Furthermore, the same cargo often uses both types of cytoskeletal tracks for the movement. This remarkable complexity of transport mechanisms requires precise regulation to ensure delivery to specific cellular destinations. Here, we propose to examine the mechanisms of regulation of intracellular transport using Xenopus melanophores as an experimental system. The major function of these cells is fast and synchronous redistribution of thousands of membrane-bounded pigment granules, which aggregate in the cell center or uniformly disperse throughout the cytoplasm by moving along MTs and AFs by means of cytoplasmic dynein (aggregation), and kinesin-2 and myosin Va (dispersion). Switching between these motors and cytoskeletal systems is regulated by a single second messenger, cAMP, and involves the activity of Protein Kinase A (PKA). Background data by the PI indicate that changes in properties of MTs and AFs may play active role in regulation of pigment granule transport. Preliminary data also indicate that PKA is bound to pigment granules where it forms complexes with molecular motors that may include other protein kinases and phosphatases regulating motor activities. This proposal will use a combination of molecular, cellular, and biochemical approaches to test the hypothesis that aggregation and dispersion signals control redistribution of pigment granules through cooperation of two interdependent mechanisms, regulation cytoskeletal tracks that provide rails for the movement, and molecular motors that generate force for transport. Specific Aims of this grant application are: (1) To test the hypothesis that transport of pigment granules is regulated by changes in availability and transport properties of MT and AF tracks; (2) To test the hypothesis that regulation of pigment granule motility involves localized signaling molecules bound to pigment granules that form complexes with molecular motors and control their activities.